Selective activation of Gαob by an adenosine A1 receptor agonist elicits analgesia without cardiorespiratory depression

The development of therapeutic agonists for G protein-coupled receptors (GPCRs) is hampered by the propensity of GPCRs to couple to multiple intracellular signalling pathways. This promiscuous coupling leads to numerous downstream cellular effects, some of which are therapeutically undesirable. This is especially the case for adenosine A1 receptors (A1Rs) whose clinical potential is undermined by the sedation and cardiorespiratory depression caused by conventional agonists. We have discovered that the A1R-selective agonist, benzyloxy-cyclopentyladenosine (BnOCPA), is a potent and powerful analgesic but does not cause sedation, bradycardia, hypotension or respiratory depression. This unprecedented discrimination between native A1Rs arises from BnOCPA’s unique and exquisitely selective activation of Gob among the six Gαi/o subtypes, and in the absence of β-arrestin recruitment. BnOCPA thus demonstrates a highly-specific Gα-selective activation of the native A1R, sheds new light on GPCR signalling, and reveals new possibilities for the development of novel therapeutics based on the far-reaching concept of selective Gα agonism.


Supplementary Fig. 2. BnOCPA, but not the A3R agonist 2-Cl-IB-MECA, selectively inhibits membrane hyperpolarisation induced by prototypical A1R agonists.
A, Membrane potential trace recorded from a CA1 pyramidal cell. BnOCPA (300 nM) reduced the effect of CPA (300 nM; quantified in main text Fig. 1i). B, The same solution of BnOCPA (300 nM), which had no effect on membrane potential, abolished synaptic transmission in a sister slice (inhibition fitted with a single exponential;  = 2. Interactions were detected via BRET using a C-terminally Nluc-tagged GPCR (A1R, left panels, or A3R, right panels) and C-terminally YFP-tagged β-arrestin1 or β-arrestin2, or pcDNA3.1 (negative control). Cells were stimulated with 3 agonists (top panels -adenosine; middle panels -BnOCPA; lower left -CPA; lower right -NECA). Note lack of either β-arrestin1 or β-arrestin2 recruitment to the A1R either by adenosine, CPA or BnOCPA, which yield BRET signals comparable to the vector control experiments (pcDNA3.1; top panels). A3R recruitment of β-arrestin2 is provided as a positive control for the BRET assay. Data are presented as mean values ± SEM from 4 individual replicates in each condition. Supplementary Fig. 4. GRK dependence of β-arrestin1 or β-arrestin2 recruitment to the hA1R.
A, Human G protein receptor kinase (hGRK) isoforms 1 -6 were over expressed (5-fold relative to A1R-Nluc), in the presence of control vector (pcDNA3.1) or β-arrestin1-YFP or β-arrestin2-YFP. BRET coupling was examined for each of these combinations for adenosine (left panels); BnOCPA (middle panels) or CPA (right panels). Data are presented as mean values ± SEM from 4-6 individual replicates in each condition. B, Heat map describing the peak maximum β-arrestin1 (left panel) and β-arrestin2 (right panel) recruitment for hA1R in the presence of the 6 GRK isoforms. A3R β-arrestin recruitment is included as a control. In all cases minimal β-arrestin recruitment was observed for the three agonists at the A1R. The ability of adenosine, BnOCPA, CPA and NECA to activate each individual Gi/o/z subtype was determined in CHO-K1-hA1R cells, transfected with PTX-insensitive G proteins or control (pcDNA3.1). cAMP levels were measured following 30 minute co-stimulation with 100 nM forskolin and each agonist. Adenosine displayed an ability to inhibit cAMP production via activation of Gi2, Goa, Gob, and Gz; CPA and NECA via Gi2, Goa and Gob, and BnOCPA exclusively via Gob. Data represented as the average level of cAMP production relative to that observed upon stimulation with 100 nM forskolin, ± SEM, of n = 4 -6 individual replicates. Stimulation of cAMP production reflects activation of endogenous Gs by the A1R and is in agreement with previous observations 1-3 .

Supplementary Fig. 7 TRUPATH assays of Goa and Gob activation and the influence of interfering peptides against Goa and Gob.
A Concentration-response curves (from 6 -8 biological replicates performed in duplicate) for the agonist-induced dissociation of Gα and Gβγ subunits in the TRUPATH BRET assay for Goa and Gob activation. Ratios have been baseline corrected with respect to a blank sample. Data are presented as mean values ± SEM. B Effects of increasing doses (in ng of plasmid) of interfering and scrambled peptides on the BRET ratio obtained from Goa and Gob in response to 1 μM CPA (4 biological replicates performed in duplicate). Inhibition of the CPA-induced BRET signal is only seen when the interfering peptide is used against its cognate Go isoform. The scrambled Goa peptide has no effect on the CPA-induced BRET signal induced by either Goa or Gob. Individual data points are presented, with the bar chart representing mean values ± SEM.
The root mean square deviation (RMSD) was computed with respect to the A1R inactive conformation. Compared to the inactive conformation (grey), in the active state (orange) the distal portion of TM7 is moved towards the TM bundle core (which is responsible for G protein binding). Starting from the active conformation (orange) and in absence of bound G protein, simulations should allow the structure to partially relax towards the inactive state (grey) with a dynamic influenced by the orthosteric ligand.
Supplementary Table 3. Transient hydrogen bonds between α4-β6 loop residue 317 (N317 in Goa, H317 in Gob), the α3-β5 loop residue D263, and the residue on H8 of the A1R (Ballesteros Weinstein enumeration in superscript). reduced heart rate (HR) from 41.8 ± 1.3 BPM to 35.5 ± 1.3 BPM. BnOCPA (300 nM) had no effect on HR (42.8 ± 1.2 BPM vs 42.1 ± 1.2 BPM; change 0.6 ± 0.2 BPM), an effect that was significantly different from that of adenosine (blue line; P = 2.22 x 10 -5 ). BnOCPA significantly (cyan line; P = 1.31 x 10 -5 ) reduced the effects of subsequent adenosine applications (from a reduction of 6.3 ± 0.6 BPM to 0.3 ± 0.2 BPM). CPA (300 nM) reduced HR by 6.1 ± 0.1 BPM, a value similar to that of adenosine. One way ANOVA on the difference in HR across the 4 conditions (F(3,9) = 64.64; P = 2.070 x 10 -6 ), with the reported Bonferroni-corrected P values. Individual data points are presented, with the bar chart representing mean values ± SEM. B, Representative traces from a urethane-anaesthetised, spontaneously breathing rat. BnOCPA blocks the effect of adenosine on heart rate (left traces), but only prevents the early phase of adenosine-induced hypotension (right trace). Data taken from the trace in Fig. 5. Scale bars measure 100 BPM or 20 mm Hg and 6 s. C, Data summary for 3 urethaneanaesthetised, spontaneously breathing rats. Bar charts showing that injection of 0.9 % saline (equivalent volume to drug experiments) had no effect (two-tailed paired t-test) on either HR (P = 1) or mean arterial blood pressure (MAP; P = 0.422). D, Data summary for 3 urethane-anaesthetised, spontaneously breathing rats. Repeated adenosine injections have the same significant effect on HR (P = 1.40 x 10 -4 and 1.02 x 10 -4 , respectively) and MAP (P = 0.012 and 0.008, respectively) and thus show no run down. One-way RM ANOVA for both HR (Greenhouse-Geisser corrected F(1.97,3.94) = 96.79, P = 4.48 x 10 -4 , and MAP (F(1.10,2.20) = 19.46, P = 0.040) from 3 animals. In C and D, each symbol represents data from a single rat with the bar chart representing mean values ± SEM. ns, not significant; , P < 0.02; , P < 0.001; , P < 0.0001. Supplementary Fig. 11. BnOCPA has no actions on cardiorespiratory parameters, but antagonizes the effects of CPA.

Coupling Systems
Examples of traces from a single spontaneously breathing urethane-anaesthetised rat showing: blood pressure (BP), from which heart rate (HR), and mean arterial pressure (MAP) are calculated, and tracheal tube airflow, from which respiratory frequency (Freq), tidal volume (VT) and minute ventilation (VE) are calculated. Applications of CPA (6 µg/kg; red vertical line), BnOCPA (8 µg/kg, 17 µg/kg, and 25 µg/kg; blue vertical lines), and the coapplication (purple vertical line) of BnOCPA (8 µg/kg) and CPA (6 µg/kg) are shown by the vertical lines. BnOCPA and CPA were given as a 350 µL/kg IV bolus. The intravenous cannula was flushed with 0.9% saline to remove compounds in the tubing between drug applications. The second phase of the blood pressure response following the first dose of CPA is likely the result of the hyponea.